This proposal is focused on developing new approaches and strategies to the discovery of metalloprotein inhibitors. Metalloproteins are an important class of medicinal targets that are relevant to treating numerous diseases including cancer, arthritis, bacterial infections, and many others. Most therapeutics that inhibit metalloproteins use a metal-binding group (MBG) to bind to the active site metal ion. Despite the importance of these MBGs, very few studies have developed methods to identify, optimize, and understand what MBGs are best for a given metalloprotein of interest. This proposal seeks to answer the 'what, why, and how' about the MBGs used in metalloprotein inhibitors, namely: a) WHAT MBGs best inhibit a given metalloprotein? b) WHY some MBGs give the best inhibition against a certain metalloprotein? c) HOW can the discovery, evaluation, and development of MBGs be accelerated from hit-to-lead? Answering these questions will address a fundamental problem at the intersection of medicinal and bioinorganic chemistry. Our research group is experienced in the field of metalloprotein inhibitors, and we have made substantial progress toward answering the aforementioned questions. Fundamentally, our laboratory is pursuing a fragment-based lead discovery (FBLD) approach to inhibitor development, where for the first time, metal chelators are being used as fragments that will explore chemical space targeted to binding metalloprotein active sites. In implementing this approach we have developed a chelator-fragment library (CFL) that can be readily screened against metalloproteins to identify what MBGs bind best to a given target. We have also determined the structure of an MBG in the active site of a metalloprotein to understand why the MBG binds with high affinity to the active site. Next, we have used and combination of bioinorganic model complexes and computational docking approaches to develop improved methods to determine how the discovery of MBG fragments can be developed into lead inhibitors. Finally, we have combined all of the aforementioned studies to prepare chelator sublibraries that provide more advanced fragment hits that probe beyond the metal ion active site. In our ongoing studies we will screen our libraries against a large number of metalloproteins (Aim 1, Aim 4), but will focus our more detailed studies on two specific Zn2+dependent systems (Aim 2, Aim 3), the matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs). These metalloproteins, which share a common metal active site motif, but different MBG preferences, will serve as excellent systems against which to test our ideas and compare and contrast the details of MBG binding. We propose to: 1. Develop and screen chelator fragment libraries (CFLs) against a wide range of metalloprotein targets. Using existing and new CFLs, screens against more than ten different metalloenzymes will be performed to reveal what MBGs bind best to various targets. 2. Perform structural and thermodynamic studies with proteins to obtain a molecular understanding of why a given MBG binds tightly to a given metalloprotein active site. Hits from CFLs will be examined with MMP-3 and hCAI, where: a) crystallographic structure determinations with co-crystallized MBGs will reveal key features of MBG-metalloprotein binding, and b) thermodynamic studies via isothermal titration calorimetry will provide binding constants and relevant energetic parameters. 3. Model MBG fragments in the MMP and CA active site to compare with structural data obtained from protein crystallography. Synthetic bioinorganic model compounds will be crystallized providing structural data on the unrestrained mode of MBG binding, while computational docking studies will be used to predict the overall conformation and second coordination sphere interactions between the MBG and the metalloprotein. By combining bioinorganic modeling and computational docking we will try to accurately recapitulate the crystallographic data. 4. Sublibraries containing MBG derivatives will be prepared to advance these scaffolds from hit- to-lead. Elaboration of MBG scaffolds will produce sublibraries containing compounds that can be screened against metalloproteins to provide more advanced lead structures. These will be developed for the MMPs and CAs, as well as a variety of other metalloproteins of medicinal interest.